Cancer development in humans and animals-is a multistep process involving at least two classes of genes, proto-oncogenes and tumor suppressor genes. We have shown that neoplastic transformation of Syrian hamster embryo cells (SHE) in culture is a multistep process involving both activation of proto-oncogenes and inactivation of a tumor suppressor gene. Approximately 50% of the carcinogen- induced tumor cell lines had an activated H-ras oncogene, which was a late, post-immortalization step in the neoplastic progression of these cells. We have also shown that loss or inactivation of tumor suppressor genes is an essential step in the multistep neoplastic transformation of SHE cells. Nontumorigenic variants have been isolated which have lost (sup-) or retained (sup+) the ability to suppress tumorigenicity of tumor cells in cell hybrids. Fusions of sup+ or sup- variants with different tumor cells show different patterns of suppression indicating that a family of tumor suppressor genes exists in these fibroblast cells. Currently, several strategies to clone tumor suppressor genes are in progress. A novel gene transfer assay has been developed using a cDNA library prepared from normal human fibroblasts in a mammalian expression vector, coupled with a highly efficient transfection procedure and a negative selection method. Another strategy involves cDNA libraries of sup+ hamster cells which have been screened with RNA from sup+ or sup- cells and differentially expressed cDNAs have been cloned. Two-dimensional gel analyses of proteins have revealed a dramatic reduction in the expression of several tropomyosin isoforms which correlate with the loss of the tumor suppressor function. A cellular phenotype associated with the loss of tumor suppressor gene function has also been found. Sup- cells suspended in agar respond reversibly to transforming and normal growth factors by forming colonies in agar whereas sup+ cells fail to grow. In addition to the tumor suppressor genes described above which are expressed in some immortal cell lines, tumorigenicity also may be limited by cellular senescence. Attempts to map the senescence phenotype to a particular human chromosome have suggested that human chromosome (#1) is involved.